the world oomycetes genetic resource collection (wogrc)
TRANSCRIPT
The World Oomycetes Genetic Resource Collection (WOGRC)formerly World Phytophthora Collection (WPC):
The history, mission, goals and projections for the future
Michael D. Coffey University of California, Riverside
Gen
us P
hyto
phth
ora
Phytophthora infestansLate Blight of Potato
(Solanaceous plants)
Phytophthora ramorumSudden Oak Death
(Ramorum Blight)
1840s – Irish potato famineDeBary (1861)
http://vassun.vassar.edu/~sttaylor/FAMINE/ILN/
Scott Bauer/ARS
http://www.msu.edu/user/douchesd/research/
COMTF
COMTF
Source: Michael D Coffey
D.C. Erwin G.A. Zentmyer
WPC founders
The origins of this important collection were in the research works of Professor Erwin and Professor Zentmyer at the University of California, Riverside
Erwin collected mainly isolates from alfalfa (lucerne)
Zentmyer isolates of P. cinnamomiand Phytophthora species from cacao
World Phytophthora Genetic Resource Collection (WPC)
Source: Michael D Coffey
D.C. Erwin G.A. Zentmyer
WPC founders
19651962
In 1962, the first accessions of the World Phytophthora Genetic Resource Collection (WPC)
were placed in glass culture tubes and a great adventure began.
The oldest deposition of the existing cultures is P0127, an isolate of Phytophthora medicaginis from
Australia
World Phytophthora Genetic Resource Collection (WPC)
There was also a limited attempt to accumulate representative species of the genus.
With Zentmyer's retirement in 1979 some of the accessions were sent to ATCC which provided them
with a core collection
Many cultures were lost at this point due to the difficulty of maintaining them using traditional methods such as
preservation under mineral oil
World Phytophthora Genetic Resource Collection (WPC)
In 1986 a major development was the provision of funds by the UC Genetic Resources Conservation Program
(UC GRCP) for Imperiled Microbial Collections to allow the WPC to be stored under liquid nitrogen using
cryogenic techniques
UC GRCP was terminated in June 2008
CRYOPRESERVATION of the World Phytophthora Collection (WPC)
1981-2008
The WPC has grown in stature over the last 25 years increasing in size from 600 to over 9500 accessions of Phytophthora (August 2008) of the more than 95
species which represent our current taxonomic information on this most important oomycete genus
Many of the accessions have been intensively studied over the years and thus the WPC is not only
unique in size but also in terms of its importance as a genetic resource
World Phytophthora Genetic Resource Collection (WPC)A part of the WOGRC
This collection has grown in stature over the last 20 years increasing in size from 30 to over 900
accessions of Pythium (August 2008) representing more than 97 species.
DNA from many of the accessions has been extracted and retained in the WOGRC DNA Bank
and thus the Pythium Collection is not only unique in size but also in terms of its importance as a
genetic resource
Pythium species Genetic Resource CollectionA part of the WOGRC
95 plus species describedvariability in morphological traitslimited morphological traitsatypical isolatesisozymes, mtDNA RFLP ITS sequences
● genomics, multilocus analysis andphylogenetic species
papillate
semipapillate
nonpapillate
paragynous Iamphigynous II
paragynous IIIampigynous IV
paragynous Vamphigynous VI
palmivora
PedicelLengthand SporangialDimensions
type species v. global population
● World Phytophthora Genetic Resource Collection (WPC at UCR)
emphasis on the use of genetic traitshow many isolates need to be studied to characterize a species?what methods need to be used?
Type species versus global population
World Phytophthora Genetic Resource Collection
THE FREEZING PROCESS
Cryostorage of isolates at the WPC
For long-term storage of Phytophthora cryogenic temperatures are essential
temperatures below the glass transition temperature of water
This is the temperature at which all biological activity ceases, and is generally accepted as -130°C
Biological and chemical activity can persist as long as wateractivity exists, however below -130°C all activity ceases
World Phytophthora Genetic Resource Collection (WPC)
The basic process involves the following steps:
Equilibration of the sample with a cryoprotectant (DMSO) at room temperature
to permit uptake of the solution
Cooling of the samples at ~ 1 to 2C per min to 0C, then 10 min at OC, followed by ~1C per min
down to –10C
Following thermal equilibration of the samples prior to ice crystal growth, the temperature is then
dropped further to -44 C
Finally, the samples are cooled very rapidly from -44 C to – 120 C in ~10 min
TaylorWharton8K
Liquid orVapor-phase
THE DNA BANK
ARCHIVAL DNA SOURCES stored at -86C in ultrapure water
DNA ‘dilution’ tubes (~10ng/uL) prepared from the ARCHIVAL DNA SOURCE
These are stored at -20C in low TE
Freeze dried material stored at -70C as theFrozen Mycelium Inventory
from which to make fresh DNA extractions
Phytophthora DNA Bank
● Live cultures (~9500)● DNA Bank (~6000)● Frozen Mycelium
(~6000)● Databases (ITS
~2000)
● Molecular and Integrated Phylogeny
● Diagnostics Research● Workshops and
Training
MOLECULAR PHYLOGENY AND DIAGNOSICS
What methods need to be used?
ITS 1 and 2Β-TUBULINLARGE SUBUNIT (LSU, 28S)Cox1, Cox2
Multilocus Phylogeny
A genus-wide phylogeny for Phytophthorautilizing complete genome sequences
Evolutionary RelationshipsGenome Resources…
Phytophthora ramorum
Phytophthora sojae
Phytophthora infestans
Phytophthora capsici
Other Oomycetes (ESTs):Saprolegnia parasiticaPlasmopara halstediiAphanomyces cochlioidesHyaloperonospora parasitica
Source: Jaime Blair
Evolutionary RelationshipsAmong Phytophthora
Genome Resources…
Utilize Complete Genomes to Design Markers for Genus-wide Phylogeny
~82 + species, 200+ isolates mainly at the WPC at UC Riverside
Source: Jaime Blair
Phyt
opht
hora
Phyl
ogen
y
Marker Selection• ~225 loci identified as potentially informative• Primers designed for 27 potential markers• 16 produced PCR products, sequence data
Marker Sequencing• 1600+ sequences generated• 234 isolates representing 84 species• 7 markers chosen for phylogeny (~7600 bp)
**Amplify AND Align across genus
0.0 kb 0.5 kb 1.0 kb 1.5 kb 2.0 kbPh
ytop
htho
raPh
ylog
eny
ATG TAA60S Ribosomal Protein L10 (496 bp)
28S Ribosomal DNA (~1340 bp)
ATG TAGBeta-Tubulin (1228 bp)
ATG TAAEnolase (1253 bp)
ATG TAAElongation Factor 1 alpha (971 bp)
ATG TAAHeat Shock Protein 90 (1765 bp)
TPI G3PDHATG TAA
TigA Gene Fusion (1694 bp)
Phyt
opht
hora
Phyl
ogen
y
10 Well Supported CladesSmall Inter-Clade Distances
82 Phytophthora sp.2 Pythium outgroups
24 species not previously analyzed(including 10 new species)
ModelTest – GTR+I+GML – Garli, 1000 bootstraps with ML parametersMP – PAUP*, random addition, TBR, 1000 bootstrapsMrBayes – 2 analyses, flat priors, 2 million gen.BEAST – uniform priors, 5 million gen.
Ppseudotsugae Pcactorum
Pnicotianae Pphaseoli
Pipomoeae Pinfestans
Pquercina Pbisheriae
Pmultivesiculata Pcitricola Pspecies 22
Pbotryosa Pinflata
Ptropicalis Pmexicana Pcapsici
Pmegakarya Peronophythora litchii
Ppalmivora Parecae
Pheveae Pkatsurae
Pinundata Phumicola
Pgonapodyides Pmegasperma
Pasparagi Pnemorosa Pilicis Ppsychrophila
Pcambivora Palni Pfragariae
Puliginosa Pcinnamomi Psinensis Psojae genome
Prichardiae Perythroseptica Pcryptogea
Pkelmania Pdrechsleri
Pmedicaginis Pfoliorum Pramorum genome Pramorum
Psyringae Phibernalis
Pprimulae Pporri
Pbrassicae Pythium vexans
100
91100
100
66100
100
100
100
100
100
73100
100
49100
6499
100
99
100
100
91
73100
100
98
100
98
98
71
35
100
98
95
49
99
54
33
75
66
70
84
81
74
62
66
83
42
0.01
Clade 8
Prichardiae Perythroseptica Pcryptogea
Pkelmania Pdrechsleri
Pmedicaginis Pfoliorum Pramorum genome Pramorum
Psyringae Phibernalis
Pprimulae Pporri
Pbrassicae91100
100
49100
73100
100
98
71
54
33
75
Source: Jaime E BlairPSU
Phyt
opht
hora
Dat
abas
e
CLADE 1 STUDY
Franklin & Marshall College, Pennsylvania: Jaime E. BlairUSDA ARS Salinas: Frank Martin
Intr
aspe
cific
Div
ersi
ty Initial ITS Analysis- 219 P. infestans
(66 haplotypes)- 13 P. andina
- 3 P. ipomoeae- 3 P. mirabilis- 4 P. phaseoli
*Massy Peiman, Mike Coffey
Pinfestans P6752Pinfestans P7035Pinfestans P6747Pinfestans P6746Pinfestans P3685Pinfestans P13873Pinfestans P13841Pandina P13660Pinfestans P13626Pinfestans P13346Pinfestans P13198Pinfestans P12053Pinfestans P11633Pinfestans P10157Pinfestans P10110Pphaseoli P10145Pphaseoli P10150Pphaseoli P11082Pinfestans P12207Pandina P13539Pinfestans P12043Pinfestans P10260Pinfestans P1847Pmirabilis P10231Pinfestans P10124Pinfestans P9464Pinfestans P10052Pipomoeae (3)
Pmirabilis P3010Pmirabilis P3005Pinfestans P7722Pinfestans P12038Pinfestans P1305Pinfestans P3681Pinfestans P10112Pinfestans (8)
0.00000.00050.00100.0015
Intr
aspe
cific
Div
ersi
ty
5 “Taxa” = 5 Species?
Initial Observations:- P. sp. “andina” most likely not separate species- “P. infestans” & P. mirabilis lineage?- P. ipomoeae and P. phaseoli good species
Future Plans:- Continue sequencing - Additional isolates, recent collections
Phyt
opht
hora
Dat
abas
e
User Authentication(analysis “cart”)
Wizard, Help Menu
Data Submission(currently manually curated)
BLAST, ClustalW,Phyloviewer,Virtual RFLP
**Bongsoo Park, PSU
Phyt
opht
hora
Dat
abas
e
THE BARCODE PROJECT
Biodiversity Canada Agriculture: André Levesque, Gregg Robideau
Cytochrome Oxidase I (COI)Good primers designed that amplify a 727bp region of COINo introns in oomycete COI (as opposed to true fungi)From G.P. Robideau, A.W.A.M. de Cock, M. Peiman, K. Bala, M. D. Coffey, and C.A. Lévesque. 2008André Levesque, Gregg Robideau
78 778900
12600
Barcode<-FM85-modOom-COI-Lev-up->
Martin, F. N., and P. W. Tooley. 2003. Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes. Mycologia 95:269-284.
contiguous Pythium
globose Pythium
filamentous Pythium
Pythium Clade K Phytopythium
Phytophthora
Pythium Clade KPythium Clade K
Pythium insidiosum
Phytopythium gen. nov.
contiguous Pythium
globose Pythium
filamentous Pythium
Phytophthora (WPC)
WORKSHOPS
In 22 years the number of accessions in the WPC has grown to ~9500 representing over 95 species. Also
represented are accessions representing the 15 described species within the marine genus
Halophytophthora.
In the last 12 years a concerted effort has been made to add a large collection of Pythium species to the
cryostorage inventory. In the last two years this effort has grown exponentially and there are now over 900 accessions representing 97 species of Pythium
An initial effort has been made to rescue the remnants of the Michael W. Dick Aquatic Phycomycetes Collection
(APCC) now held at CABI.
The original mission of the World PhytophthoraCollection (WPC) has been broadened with the
accumulation of the phylogenetically comprehensive Pythium Collection and will continue with the
acquisition of additional genera within the Kingdom Straminipila.
The new name for this collection will be the World Oomycetes Genetic Resource Collection
(WOGRC).
The future goals of the WOGRC are threefold:
1) maintenance and expansion of a worldwide collection of genera within the Kingdom Straminipila
2) development of a DNA Bank to provide DNA for research
3) creation of online databases providing phenotype and
genotype information on important genera.
2006
Postdoctoral scholars: Masoomeh Peiman, Tatiana Roubtsova, Alexei KravtsovLab Assistants: Avneet Brar, Iona Cunningham, Sandra VerdinUndergraduate researchers: Julie Huss, Thomas Vu,
Bharat Sunkavally, Shirley Tu, Erik Haw, Elaine XuJustin Chao, Shinly Du, Daniel Guindi, Carrie Tran, Peggy Ju, Giselle Vu, Linda Vu, Kacey Cao, Serena Chai, Eric Chan, Tien Dinh, Jorge Farias, Eric Garcia, Sabrina Garrovillas, Zahra Mousavi Jasemi, Victor Kieu, Lisa Lam, Mark Luu, Octaviano Moro, Charmi Patel, Joshua Smith, Virginia Tran, Jacqueline Villanueva, Vera Wong, Candace Woo
Visiting scientists: Byung-Sup Kim, Grazyna Szkuta, Laura Gaggero, Pedro Martin, Ehab Sarhan
Collaborations!Penn State University: Seogchan Kang, Bongsoo Park, Sook-Young Park, Michele Mansfield, David GeiserFranklin & Marshall College, Pennsylvania: Jaime E. BlairUSDA ARS Salinas: Frank Martin USDA-APHIS-PPQ-PHP-PSPI-MDL: Gloria AbadUSDA-APHIS- CPHST: Phil Berger, Laurene LevyUSDA ARS Corvallis: Niklaus GrunwaldBiodiversity Canada Agriculture: André Levesque, Gregg RobideauCBS Utrecht, The Netherlands: Arthur de CockCSL, York, UK: Kelvin HughesOhio State University– Carla Garzon and Sophien Kamoun (Sainsbury Lab, UK) IFAPA Spain: Rosa M. Pérez Jiménez, Pedro MartinUC Riverside: Hailing Jin, Greg DouhanCIAT Colombia: Elizabeth AlvarezUniversidad de los Andes Colombia: Silvia RestrepoCIP Peru and Ecuador: Greg ForbesMSU and VNIIF: Russia: Sergey Elansky, Alexei Filippov India NRRI: Bindu Roy C.Vietnam and Australia: Doan Nhan Ai, David Guest, Andre Drenth
THE END